The mechanism(s) of breast cancer progression to steroid receptor (SR)-positive but hormone refractory breast cancer remain elusive. Much of the research in this field is focused on the linkage of estrogen receptor-? (ER) to signal transduction, as mediated by peptide growth factor activation of tyrosine kinase receptors; these molecules are important clinical targets. However, progesterone receptors (PR) may also play a key role as mediators of early breast cancer progression. Recently, we uncovered a novel mechanism of hormone-independent hyperactivation of PR transcriptional activity that is mediated by membrane-initiated phosphorylation events. In response to mitogenic protein kinases (c-Src, MAPKs, CDK2) often elevated in breast cancer, persistent phosphorylation of PR Ser294 blocks ligand-induced sumoylation at K388 (a repressive modification). Abundant total PR is a marker of good tumor behavior. However, we hypothesize that de-repressed phospho-PR acts on genes whose products mediate early breast cancer progression or bad tumor behavior. One check on PR hyperactivity includes rapid ligand-dependent degradation by the ubiquitin-proteasome pathway. Indeed, in the presence of hormone, phospho-PR undergoes rapid turnover, often rendering the low abundance protein undetectable by standard antibody-binding assays (i.e. clinically used IHC). Clinical observations of PR loss have led to the incorrect conclusion that PR transcriptional activity is unimportant in PR-low breast cancers. However, our studies reveal that phospho-PR is transcriptionally hyperactive and remarkably, targets genes that are not sensitive to progesterone or progestins; IRS-1 is an example of a ligand-independent phospho-PR gene. Furthermore, under conditions of high kinase activities, Ser294-phosphorylated PRs are not sumoylated, and thus fail to transrepress ER but may instead cooperate with ER at non-classical (non-PRE containing) gene targets in the complete absence of steroid hormones; STC-1 is an example of an ER-gene that is de-repressed by phospho-PR. Notably, the proliferation of breast cancer cell models expressing mutant PRs that cannot be sumoylated (K388R) is very sensitive to estrogen, but resistant to anti-estrogen. Similarly, we predict that phospho-PR signaling drives the growth of some hormone-refractory breast cancers that can be identified by a unique PR gene signature. Herein, we will define the phospho-PR gene signature using unique cell line models engineered for inducible PR expression. The role of the PR sumoylation/phosphorylation switch in the regulation of hormone responsiveness at endogenous genes will be defined (Mechanisms; Aim 1). Our unique phospho-PR gene signature will be validated in in vitro models of tam- and AI- (aromatase inhibitor) resistance and in human tumors (Validation; Aim 2). Finally, our innovative hypothesis will be tested in AIB1-transgenic mice, an in vivo model of SR-driven (ER+/PR+) and tam-resistant breast cancer (Biology; Aim 3). Little is known about how PR and ER/PR interactions contribute to hormone resistance in breast tumors. Our studies on phospho- PR signaling will provide valuable insight into the coordinated regulation of PR and ER function by pathways that can be easily targeted for therapeutic intervention.